A new approach to insect-pest control—combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity

Voltage-sensitive sodium channels are responsible for the generation of electrical signals in most excitable tissues and serve as specific targets for many neurotoxins. At least seven distinct classes of neurotoxins have been designated on the basis of physiological activity and competitive binding studies. Although the characterization of the neurotoxin receptor sites was predominantly performed using vertebrate excitable preparations, insect neuronal membranes were shown to possess similar receptor sites. We have demonstrated that the two mutually competing antiinsect excitatory and depressant scorpion toxins, previously suggested to occupy the same receptor site, bind to two distinct receptors on insect sodium channels. The latter provides a new approach to their combined use in insect control strategy. Although the sodium channel receptor sites are topologically separated, there are strong allosteric interactions among them. We have shown that the lipid-soluble sodium channel activators, veratridine and brevetoxin, reveal divergent allosteric modulation on scorpion α-toxins binding at homologous receptor sites on mammalian and insect sodium channels. The differences suggest a functionally important structural distinction between these channel subtypes. The differential allosteric modulation may provide a new approach to increase selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective toxins. Thus, a comparative study of neurotoxin receptor sites on mammalian and invertebrate sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins, and may offer new targets and approaches to the development of highly selective pesticides.     

Modulation of neuronal voltage-gated calcium channels by farnesol.

The modulation of presynaptic voltage-dependent calcium channels by classical second messenger molecules such as protein kinase C and G protein betagamma subunits is well established and considered a key factor for the regulation of neurotransmitter release. However, little is known of other endogenous mechanisms that control the activity of these channels. Here, we demonstrate a unique modulation of N-type calcium channels by farnesol, a dephosphorylated intermediate of the mammalian mevalonate pathway. At micromolar concentrations, farnesol acts as a relatively non-discriminatory rapid open channel blocker of all types of high voltage-activated calcium channels, with a mild specificity for L-type channels. However, at 250 nM, farnesol induces an N-type channel-specific hyperpolarizing shift in channel availability that results in approximately 50% inhibition at a typical neuronal resting potential. Additional experiments demonstrated the presence of farnesol in the brain (rodents and humans) at physiologically relevant concentrations (100-800 pmol/g (wet weight)). Altogether, our results indicate that farnesol is a selective, high affinity inhibitor of N-type Ca(2+) channels and raise the possibility that endogenous farnesol and the mevalonate pathway are implicated in neurotransmitter release through regulation of presynaptic voltage-gated Ca(2+) channels.     

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

N-type voltage-gated calcium channels localize to?presynaptic nerve terminals and mediate key events?including synaptogenesis and neurotransmission.?While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α(1) subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic function.     

Calcium-induced modulation of synaptic transmission

Calcium (Ca²⁺) is a second messenger regulating a wide variety of intracellular processes. Using GABA-and glycinergic synapses as examples, this review analyzes two functions of this unique ion: postsynaptic Ca²⁺-dependent modulation of receptor-operated channels and Ca²⁺-induced retrograde regulation of neurotransmitter release from the presynaptic terminals. Phosphorylation, rapid Ca²⁺-induced modulation via intermediate Ca²⁺-binding proteins, and changes in the number of functional receptors represent the main pathways of short-and long-term plasticity of postsynaptic receptor-operated channel machinery. Retrograde signaling is an example of synaptic modulation triggered by stimulation of postsynaptic cells and mediated via regulation of presynaptic neurotransmitter release. This mechanism provides postsynaptic neurons with efficient tools to control the presynaptic afferents in an activity-dependent mode. Elevation of intracellular Ca²⁺ in a postsynaptic neuron triggers the synthesis of endocannabinoids (derivatives of arachidonic acid). Their retrograde diffusion through the synaptic cleft and consequent activation of presynaptic G-protein coupled to CB1 receptors inhibits the release of neurotransmitter. These mechanisms of double modulation, which include control over the function of postsynaptic ion channels and retrograde suppression of the release machinery, play an important role in Ca²⁺-dependent control of the main excitatory and inhibitory synaptic pathways in the mammalian nervous system.     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

Neuronal sodium channels in ventricular heart cells are localized near T-tubules openings

Cardiac voltage-dependent sodium channels (VDSC) are known to be tetrodotoxin (TTX)-resistant. However, recent immunochemical studies suggest the presence of TTX-sensitive neuronal-type VDSC in the heart. Scanning ion conductance microscopy (SICM) coupled to electrophysiology was used to obtain more direct functional evidence. TTX sensitivities of whole-cell sodium currents (I(Na)) in control and detubulated cells were compared. Addition of 200 nM TTX decreased I(Na) of control cells by 20%, whereas detubulated cells were hardly effected. The remaining current peaked slightly earlier and inactivation decay was faster (as in neuronal VDSC) than in detubulated cells. Single-channel activity was first assayed at random on the plasmalemma, and after topography had been revealed by SICM, at patched T-tubules openings. In the latter case, a single-channel conductance of 11-12pS was observed with a higher rate of success. This study provides independent evidence for neuronal VDSC in cardiomyocytes where they could rapidly and synchronously couple T-tubule and cell surface depolarizations.     

Regulation of Synaptic Transmission by Presynaptic CaMKII and BK Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the BK channel are enriched at the presynaptic nerve terminal, where CaMKII associates with synaptic vesicles whereas the BK channel colocalizes with voltage-sensitive Ca²⁺ channels in the plasma membrane. Mounting evidence suggests that these two proteins play important roles in controlling neurotransmitter release. Presynaptic BK channels primarily serve as a negative regulator of neurotransmitter release. In contrast, presynaptic CaMKII either enhances or inhibits neurotransmitter release and synaptic plasticity depending on experimental or physiological conditions and properties of specific synapses. The different functions of presynaptic CaMKII appear to be mediated by distinct downstream proteins, including the BK channel.     

Calcium channels involved in neurotransmitter release at adult,neonatal and P/Q-type deficient neuromuscular junctions (Review)

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (I Ca ) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca 2+ inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca 2+ -inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.     

Calcium channels involved in neurotransmitter release at adult,neonatal and P/Q-type deficient neuromuscular junctions (Review)

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (Ica) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca(2+) inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca(2+)-inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.     

神经递质3H-去甲肾上腺素释放与家蝇对拟除虫菊酯抗性的关系

在用K⁺去极化条件下,研究了溴氰菊酯和氯菊酯分别对敏感、抗溴氰菊酯和抗氯菊酯家蝇Musca domestica 品系脑突触体释放神经递质去甲肾上腺素的影响。结果表明：在用K⁺去极化后,神经递质去甲肾上腺素的释放在抗溴氰菊酯和抗氯菊酯家蝇品系中比敏感品系分别下降47.0%和51.0%;当用10^-5 mol/L溴氰菊酯预处理家蝇脑突触体,用K+去极化后对敏感、抗溴氰菊酯和抗氯菊酯家蝇品系释放去甲肾上腺素的加强作用分别提高80.3%、26.5%和70.5%;用10^-5 mol/L氯菊酯预处理3个家蝇品系的突触体对去甲肾上腺素释放均无加强作用。由此表明,家蝇对溴氰菊酯的抗性是与Na+通道的亲和性降低有关,而氯菊酯的抗性与Na+通道的亲和性关系不大。     

Forty Years of Sodium Channels: Structure,Function, Pharmacology,and Epilepsy

Voltage-gated sodium channels initiate action potentials in brain neurons. In the 1970s, much was known about the function of sodium channels from measurements of ionic currents using the voltage clamp method, but there was no information about the sodium channel molecules themselves. As a postdoctoral fellow and staff scientist at the National Institutes of Health, I developed neurotoxins as molecular probes of sodium channels in cultured neuroblastoma cells. During those years, Bruce Ransom and I crossed paths as members of the laboratories of Marshall Nirenberg and Philip Nelson and shared insights about sodium channels in neuroblastoma cells from my work and electrical excitability and synaptic transmission in cultured spinal cord neurons from Bruce’s pioneering electrophysiological studies. When I established my laboratory at the University of Washington in 1977, my colleagues and I used those neurotoxins to identify the protein subunits of sodium channels, purify them, and reconstitute their ion conductance activity in pure form. Subsequent studies identified the molecular basis for the main functions of sodium channels—voltage-dependent activation, rapid and selective ion conductance, and fast inactivation. Bruce Ransom and I re-connected in the 1990s, as ski buddies at the Winter Conference on Brain Research and as faculty colleagues at the University of Washington when Bruce became our founding Chair of Neurology and provided visionary leadership of that department. In the past decade my work on sodium channels has evolved into structural biology. Molecular modeling and X-ray crystallographic studies have given new views of sodium channel function at atomic resolution. Sodium channels are also the molecular targets for genetic diseases, including Dravet Syndrome, an intractable pediatric epilepsy disorder with major co-morbidities of cognitive deficit, autistic-like behaviors, and premature death that is caused by loss-of-function mutations in the brain sodium channel Na_V1.1. Our work on a mouse genetic model of this disease has shown that its multi-faceted pathophysiology and co-morbidities derive from selective loss of electrical excitability and action potential firing in GABAergic inhibitory neurons, which disinhibits neural circuits throughout the brain and leads directly to the epilepsy, premature death and complex co-morbidities of this disease. It has been rewarding for me to use our developing knowledge of sodium channels to help understand the pathophysiology and to suggest potential therapeutic approaches for this devastating childhood disease.     

Molecular mechanisms of neurotoxin action on voltage-gated sodium channels

Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.     

Effects of neurotoxins on pancreatic islets: Elucidation of a possible role for sodium channels in the secretory response

The neurotoxins veratridine and Leiurus toxin were used to characterize the nature of the sodium channel in the pancreatic β-cell membrane in relation to metabolc and secretory events. Insulin release and glycolytic flux were measured on batch-incubated rat islets. Veratridine, 200 μM, but not 10 μM, elicited a secretory response in the presence of 5.6 mM (basal) glucose, but did not influence the response to 15.3 mM glucose. Leiurus toxin, 20 nM, together with basal glucose and 10 μM veratridine induced insulin release, although Leiurus toxin, alone, was not effective. The secretory responses to the neurotoxins, but not 15.3 mM glucose, were blocked by tetrodotoxin. Glucose utilization was enhanced by 200 μM veratridine in the presence of basal glucose. Leiurus toxin at 20 nM increased the glycolytic rate which was further enhanced by the addition of 10 μM veratridine. The increments in glycolytic flux were partially or completely blocked by tetrodotoxin. Ouabain, 1.0 mM, had no effect on the secretory response to veratridine, but completely blocked the veratridine-induced increase in glycolytic flux. These observations indicate that the sodium channels in the β-cell membrane are pharmacologically similar to those in neuronal plasma membranes. Furthermore, the secretory response elicited by neurotoxins may occur independently of an increase in glycolytic flux. The major role of glycolytic flux may be to provide energy for extrusion of sodium from the β-cell.     

Translocation of botulinum neurotoxin light chain protease through the heavy chain channel

Clostridial botulinum neurotoxins (BoNTs) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. An enigmatic step in the intoxication process is the mechanism by which the neurotoxin heavy chain (HC) forms the conduit for the translocation of the light chain (LC) protease across the endosomal membrane into the cytosol, its site of action. Here we investigate the mechanism of LC translocation by using the combined detection of channel currents and substrate proteolysis, the two hallmark activities of BoNT. Our data are consistent with the translocation of the LC through the HC channel and show that the LC protease activity is retrieved in the trans compartment after translocation. We propose that the BoNT HC-LC complex embedded in the membrane is a transmembrane chaperone, a dynamic structural device that prevents aggregation and achieves translocation of the LC. In this regard, the complex is similar to the protein conducting/translocating channels of the endoplasmic reticulum, mitochondria and chloroplasts.     

Glycerotoxin stimulates neurotransmitter release from N-type Ca2+ channel expressing neurons

Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular junction by directly interacting with N-type Ca2+ (Cav2.2) channels. Here we have utilized GLTx as a tool to investigate the functionality of Cav2.2 channels in various mammalian neuronal preparations. We first adapted a fluorescent-based high-throughput assay to monitor glutamate release from rat cortical synaptosomes. GLTx potently stimulates glutamate secretion and Ca2+ influx in synaptosomes with an EC50 of 50 pm. Both these effects were prevented using selective Cav2.2 channel blockers suggesting the functional involvement of Cav2.2 channels in mediating glutamate release in this system. We further show that both Cav2.1 (P/Q-type) and Cav2.2 channels contribute equally to depolarization-induced glutamate release. We then investigated the functionality of Cav2.2 channels at the neonatal rat neuromuscular junction. GLTx enhances both spontaneous and evoked neurotransmitter release causing a significant increase in the frequency of postsynaptic action potentials. These effects were blocked by specific Cav2.2 channel blockers demonstrating that either GLTx or its derivatives could be used to selectively enhance the neurotransmitter release from Cav2.2-expressing mammalian neurons.     

Inhibition of Catecholamine Secretion from Adrenal Medulla Cells by Neurotoxins and Cholinergic Antagonists

Abstract: The effects of several neurotoxins and cholinergic antagonists on the nicotine-induced secretion of catecholamines by adrenal medulla cells in culture were investigated. Aconitine, veratridine, and batrachotoxin, in the presence of 1 μ m -tetrodotoxin inhibited the nicotine-stimulated secretion of catecholamines in a dose-dependent manner in Locke's solution. In Na⁺-free sucrose medium, tetrodotoxin was not required to inhibit the stimulatory effects of aconitine, veratridine, and batrachotoxin, and these agents by themselves inhibited the nicotine-stimulated secretion of catecholamines. Scorpion venom, which also increases the flux of Na⁺ through tetrodotoxin-sensitive channels, was not an effective inhibitor of nicotine-stimulated secretion. Histrionicotoxin, atropine, hexamethonium, and decamethoniun–as well as the Na⁺-channel activators–noncompetitively inhibit nicotine-stimulated secretion. The effects of these agents on nicotine-stimulated secretion appear similar to their effects on the inhibition of depolarization at the neuromuscular junction. Reversibility studies suggest that the stimulatory and inhibitory sites of the neurotoxins are different, while studies in Na⁺-free media suggest that tetrodotoxin-insensitive sodium channels are not involved in the inhibitory effect of the neurotoxins. A possible site of action for the inhibitory effects of the neurotoxins. A possible site of action for the inhibitory effects of the neurotoxins is the nicotinic-receptor-associated ion channel.     

钠离子通道疾病及其抑制剂生物学功能研究进展

电压门控型钠离子通道(Voltage-gated sodium channel,VGSC)广泛分布于兴奋性细胞,是电信号扩大和传导的主要介质,在神经细胞以及心肌细胞兴奋传导等方面发挥重要作用。钠离子通道结构和功能的异常会改变细胞的兴奋性,从而导致多种疾病的发生,如神经性疼痛、癫痫,以及心律失常等。目前临床上多采用钠离子通道抑制剂治疗上述疾病。近些年,研究人员陆续从动物的毒液中分离纯化出具有调控钠离子通道功能的神经毒素。这些神经毒素多为化合物或小分子多肽。现已有医药研发公司将这些天然的神经毒素进行定向设计改造成钠离子通道靶向药物用于临床疾病的治疗。此外,来源于七鳃鳗Lampetra japonica口腔腺的富含半胱氨酸分泌蛋白(Cysteine-rich buccal gland protein,CRBGP)也首次被证明能够抑制海马神经元和背根神经元的钠离子电流。以下针对钠离子通道疾病及其抑制剂生物学功能的最新研究进展进行分析归纳。     

Structure and function of voltage-gated sodium and calcium channels.

The primary structures of the Na+ channel alpha-subunits from several species have now been deduced from cDNA sequences, and complete primary structures of all of the subunits of skeletal muscle L-type Ca2+ channels have been defined. Current research on voltage-gated ion channels is focusing on defining the structural components responsible for specific aspects of channel function. Recent experiments have identified regions of these channels that are important for voltage-dependent activation and inactivation, ion conductance, regulation by protein phosphorylation, and modulation by drugs and neurotoxins using a combination of antibody mapping and site-directed mutagenesis approaches. The results form the outlines of a structural map of ion channel function.     

Depolarization Differentially Affects Allosteric Modulation by Neurotoxins of Scorpion α-Toxin Binding on Voltage-Gated Sodium Channels

Abstract: Voltage-gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K⁺ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion α-toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 m M K⁺). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 m M K⁺). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two-state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state-dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.